Excellent progress has been made over the past twenty years in the adaptation of attenuated bacterial vaccine strains for expression of foreign antigens to create multivalent live vector vaccines. This has included a devotion of significant effort to the creation of expression technologies which either directly or indirectly address the important problem of metabolic stress often associated with expression of foreign immunogens.[1,2] It is recognized that inappropriate synthesis of high levels of foreign protein in an effort to induce an antigen-specific protective immune response can adversely affect the fitness and growth rate of an already attenuated vaccine strain, resulting in over-attenuation and loss of immunity directed at both the live vector and foreign antigen. If these target immunogens are encoded by multicopy expression plasmids, these undesirable metabolic fluxes can result in plasmid loss in the absence of selective pressure, which ultimately defeats the strategy of live vector-mediated delivery of vaccine antigens.
Effective genetic stabilization systems have been developed for enhancing the retention of multicopy plasmids encoding regulated synthesis of foreign antigens, without the further requirement to select with antibiotics.[3,4,5] Antigen export systems have also been developed to reduce proteolytic degradation of foreign antigens within the cytoplasm and more effectively deliver these antigens to the immune system to enhance immunogenicity.[6,7,8,9] Thus, a variety of genetic techniques and technologies are now available for efficient delivery of one or more antigens using live vector vaccines. However, significant problems remain associated with this technology. For example inclusion of more than one gene encoding a foreign antigen of interest within a single multicopy plasmid can lead to large plasmids which ultimately prove to be genetically unstable, reducing both antigen synthesis and the ensuing immune responses.[10]
Novel strategies for engineering live vector vaccines to express high levels of a foreign antigen or to express two or more different antigens are needed.